Hepatitic C virus (HCV) is the single member of the genus hepacivirus in the family Flaviviridae. The genome consists of a single positive-stranded RNA molecule. The HCV genome differs from many other RNA viruses in that it possess neither a cap structure not a poly (A)-tail. The recognition of the positive and negative strand copies of the viral genome by the replication complex required an extraordinary level of specificity. The template specificity is thought to occur through cis-acting RNA signals located at the termini of the viral genome, although internally located replication signals have also been identified. The 5' NTR of several viruses has now been shown to be involved in translation, minus-strand, and plus-strand synthesis. The overall goal of this proposal is to determine which RNA sequence, and structural elements, located in the 5' end of the viral genome are involved in HCV RNA synthesis and determine if these signals play a role in plus- or minus-strand synthesis. We hypothesize that the 5' NTR and the C-E1-E2-NS2 region of the HCV open-reading frame contain elements that are essential, for modulating HCV RNA synthesis. We will use a modified replicon system that separates the translational functions of the 5' NTR from its role in replication. A systematic mutational analysis will be performed to localize regions in the 5' NTR that affect replication efficiency. The regions in the C-E1-E2-NS2 coding sequence that modulate replication will be delineated by a series of deletion mutants. Positive-strand accumulation will be detected by luciferase expression and negative strand levels will be determined by quantitative RT-PCR. The level and ratio of these two RNAs will be analyzed as an indicator to the step at which the impairment of replication occurs. We will design specific mutations and utilize modified anti-sense oligo nucleotides to specifically block RNA elements in either plus- or minus-strand orientation. The results will lead to a better understanding of the signals located at the 5' end of the genome involved in template specificity and can lead to the development of more efficient full length replicon systems and will aid the development of compounds that interfere with the formation of replication signals.